实验动物总则

Hepatitis B virus (HBV) is the human member in the family Hepadnaviridae. Several bird and rodent hepadnaviruses have been isolated, including duck (1), heron (2), woodchuck (3), ground squirrel (4), and Arctic ground squirrel HBVs (5). A primate hepadnavirus has recently been isolated from a New ...

Hepadnaviruses are small, DNA-containing viruses that replicate by reverse transcription (1). They have a lipid envelope surrounding an icosahedral protein core particle, whose shell is composed of a single viral protein, the core protein. Within the core particle, the viral reverse tr ...

Hepatitis B virus (HBV) transcriptional regulation has been extensively studied in vitro by transient transfection using the activity of a reporter molecule as the readout for transcriptional activity (1–5). Several reporter molecules, such as chloramphenicol acetyl transfe ...

In vitro systems, providing a defined and controllable chemical environment, have been instrumental in understanding the replication properties of many viruses. A classic example is simian virus 40 (SV40), which, in conjunction with its T antigen, uses host-encoded DNA polymerase for c ...

The aim of this chapter is to provide a succinct and integrated view of hepadnaviral infections and the broad roles of these viruses in malignant transformation in the liver. This is not an exhaustive review of hepatitis B virus (HBV) molecular biology or the literature, and as such, only selected ref ...

The continuing spread of the HIV-1 epidemic worldwide has stimulated efforts to develop vaccines and decrease transmission of HIV-1 as noted in Chapter 8. Key to this effort is the characterization of the qualities of HIV-1 in genital secretions. Seminal cell and seminal plasma culture have be ...

The ability to accurately measure viral RNA in the plasma (1–3) and intracellular (4–7) compartments of HIV-1-infected persons has led to a dramatic improvement in the understanding of the natural history of HIV-1 and AIDS. A number of recent studies have convincingly demonstrated that high le ...

Since its discovery in 1981, human immunodeficiency virus type 1 (HIV-1) has rapidly emerged as one of the most devastating infectious pathogens of this century (1–3). The World Health Organization (WHO) estimates that, as of 1995, there were at least 15 million HIV- infected men, women, and children ...

A salient feature regarding the HIV (and all the retroviruses in general) replicative cycle is the retrotranscription of the RNA genome to DNA (provirus), followed by its integration in the genome of the host cell.

The assessment of viral load in the blood, tissues, and bodily fluids of persons infected with human immunodeficiency virus is fundamental to defining the stage of disease (1–3), the effect of antiviral treatments to abate disease (4, 5), disease progression (6–8), and propensity for the transm ...

Molecular analyses of HIV-1 variation, and its impact on the biological properties of the virus have by necessity been restricted by the scarcity of cloned proviruses. Only a handful of full-length clones exist (1), and of these, even a smaller fraction are infectious upon transfection into cell ...

Genetic analysis of HIV-1 frequently involves molecular cloning. The general laboratory approach of identifying a desired molecular clone after a successful transformation includes picking colonies, growing stationary-phase bacterial cultures, isolating plasmid DNA ...

Northern blotting (1) is one of many tools used in understanding the human immunodeficiency virus type 1 (HIV-1). In the process of Northern blotting, RNA is first separated by size through a denaturing agarose gel and transferred onto a membrane. With this transfer or blotting, and subsequent hy ...

The predominant mode of transmission of HIV-1 worldwide is sexual intercourse. Therefore, there has been growing interest in studying HIV-1 in genital secretions. Given the urgency to develop a vaccine to protect against HIV-1, techniques have been developed to isolate and quantitate HIV ...

Biological membrane fusion is an important part of many cellular processes and is a critical step in the entry of enveloped viruses, such as HIV-1, into cells. For HIV-1 to infect cells, the virus must bind to the cell surface, after which the viral Env protein must be triggered to undergo a conformational c ...

HIV replication is classically measured by quantitating virus present in the supernatant of infected cells over time. Typically, cells are infected at low multiplicity of infection (MOI) and washed extensively to remove input virus. Samples of culture supernatant are then removed over ...

CD4 was identified in 1984 as the receptor for HIV-1 (1,2). However, it was soon apparent that a second receptor was necessary for HIV-1 infection of CD4+ cells. This coreceptor was first identified by Berger and colleagues who showed that fusion and entry of T-cell line-adapted strains of HIV-1 into CD4+ c ...

HIV-1 is routinely isolated by cocultivation of patient PBMC with mitogen-stimulated HIV-uninfected donor PBMC (see Chapter 1). In this culture system, HIV-1 primarily replicates in CD4+ T-lymphocytes, and such viruses are termed clinical or primary isolates. As early as 1986, the in vitro r ...

This assay measures the activity of reverse transcriptase (RNA-dependent-DNA polymerase), an enzyme that synthesizes DNA using RNA as the template. The detection of reverse transcriptase activity (RT) indicates the presence of HIV-1 in in vitro cultures of human peripheral blood mon ...

This procedure is used for establishing cocultures for virus isolation by an endpoint dilution assay in a 96-well tissue culture plate. This is a cell culture assay based on the detection of HIV in patient peripheral blood mononuclear cells (PBMC) by the appearance of p24 gag protein in the culture s ...